Identification And Function Analysis Of Poplar ERF Gene In Response To Salt Stress

AP2/ERF (APETALA2/ethylene responsive factor) transcription factor family is one of the plants-specific transcription factor families. AP2/ERFs contain one or two AP2/ERF conservative domain,which is made up of about 60 highly conserved amino acids. AP2/ERF domain can directly interact with DRE/CRT cis-actingelement or GCC-box cis-acting element, and regulate the expression of many target genes. Therefore, AP2/ERFs play an important role in plant growth and development, and abiotic stress tolerance.In present study, a total of 209 poplar ERF transcription factors were identified by bioinformatic analysis. More than 82% of the 209 poplar ERF proteins contain similar and complete AP2/ERF domain, and only 8 proteins have no or incomplete AP2/ERF domain. According to the AP2/ERF domain, theses 209 PtERFs were divided into 10 groups (I-X). Group I to V belong to CBF/DREB subfamily (Group A), and group VI to X belong to ERF subfamily (Group B). Gene structure analysis showed 68 poplar ERF genes contain introns, and five of which contained more than one intron. Genes containing introns were mainly included in Group I, II, VII and VIII. Protein structure analysis revealed that 208 (except PtERF139) poplar ERF transcription factors containing motif-1 (WLG-motif), and motif-6 mainly exists in the DREB subfamily. AYD and AHDdomains are respectively existed in ERF and DREB subfamily. In addition, acid-rich regions were widely spread in theses conservative domains. RNA-seq analysis showed that 209 poplar ERF geneswere divided into three groups: lowexpression group (L), medium expression group (Ml and M2) and highexpression group (H1 and H2). Most of the highly expressed genes maily distributed in Group II, V, VI, VIII, IX.RT-qPCR analysis showed that, more than half 59 of 114 poplar ERF genes made a response to salt stress in different degrees.11 genes down-regulated, and 48 up-regulated. Among the up-regulated genes,29 up-regulated 2-4 folds,7 up-regulated 4-8 folds,4 up-regulated 8-16 folds,2 up-regulated 16-32 folds, and 6 genes up-regulated more than 32 folds. Tissue-specificexpression pattern analysis found that the expression level of poplar ERF genes in leaves was significantly higher than that in stems, and the expression level in L1 is lower than that in other locations.Similar expression patterns were found in stems, and the expression level in the top end (S1) was the lowest. There was no significant difference in the other positions. Spatial-temporal expression pattern of 13 candidate genes in response to salt stress analysis showed that, the high-expression genes in roots showed "induce-restore"expression patterns. And the expression pattern is different of different expression level genes in different tissues. PtERF136showed a significantly different expression patterns from the other genes. Its expression level in stems was significantly higher than that in other tissues. We constructed pBI121-PtERF3, pBI121-PtERF37, pBI121-PtERF136 and pBI121-PtERF159 plant expression vectors, and planted transgenic Arabidopsis.Resistance analysis under salt stress conditions showed that the transformation ofPtERF genes can significantly improve the salt tolerance of transgenic Arabidopsis. The result indicating that ERF genes play an important role in plant resistant to adversity stresses. Subcellular localization analysis with pUC18-35S-PtERF3-GFP, pUC18-35S-PtERF37-GFP, pUC18-35S-PtERF136-GFP and pUC18-35S-PtERF159-GFP recombinant plasmids by onion epidermal gene gun instantaneous conversion technology showed that, PtERF3, PtERF136 and PtERF159were localized in the cell membrane, cytoplasm and nucleus, and PtERF37was localized in the nucleus.RNA sequencing analysis of PtERF37 transgenic Arabidopsis showed that, PtERF37 improved plant salt stress toleranceby regulated the expression of some endogenous abiotic stress related genes,such as AT3G04720.1, AT1G73260.1,AT3G05640.1, AT5G15230.1, AT1G06160.1, AT5G44420.1, AT3G04720.1, AT2G26020.1, etc. All of this provides an important theory basis for breeding new resistant plant species using this ERF gene.